A PROCESS FOR THE PREPARATION OF A NOVEL BIO-CURD

Abstract

The invention gives a process for the preparation of shelf-stable probiotic curd using a locally isolated strain of Pediococcus pentosaceus. This has been deposited at Microbial type culture collection center, Chandigarh with an Accession No. MTCC 5151. The probiotic culture has shown a good shelf-life till 21 days where it maintains the viable cell count as prescribed by WHO. The culture has antimicrobial activity against the toxic food-pathogen, Staphylococcus aureus and has probiotic properties like resistance to low acid and high bile salt mix. This makes the probiotic curd a novel healthful food product for societal benefit.

Full Text

The present invention relates to a process for the preparation of shelf stable probiotic curd. The main utility of the invention is that this particular locally isolated food grade microorganism, which is probiotic, has shown excellent shelf life in curd as a suitable carrier. This has shown the importance of this particular culture in the preparation of a novel probiotic curd to provide potential health benefits to the consumers. A sample of strain has been deposited during February 2004 in the Culture Collection Centre under the Accession No. MTCC 5151.
Probiotics are live microorganisms and play very important role in the gut region. Today, the consumers have a strong inclination towards natural food items for health benefits instead of medicines. Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bact, 66: 365-378, have shown that lactic acid bacteria are highly important in providing protection to humans against various intestinal tract infections. It has been shown earlier (Haenel,H. and Bendig,J. 1975. Intestinal flora in health and disease progress. Food Nutr. Sci., 1: 21-64) that these bacteria protect against food pathogens and also improves the immune system (Kalliomaki,M., Salminen,S., Arvilommi,H., Kero,P. and Isolauri,E. 2001. Probiotics in primary prevention of atopic disease: a randomized placebo controlled trial. Lancet, 357:1076-

1079). Health benefits have been shown by consumption of cultured milk and other products containing lactic acid bacteria.
As interest the link between diet and health has gathered pace and consumers feel well and stay healthy by eating nutritionally designed foods. Today, there is a market for diverse foods that assist in constructing a healthy diet (Pimia R.Pupponen, Aura,A.M., Oksman,K.M., Caldentey,P., Myllarinen,M., Saarela., Sandholm,J.M. and Poutanen,K. 2002. Trends in Food Sci. Technol., 13: 3-11). When specific functional ingredients are used its stability and viability is important. There is a growing scientific evidence to support the concept that the maintenance of a healthy gut microflora may provide protection against gastro-intestinai disorders and even cancer (Mitsouka,T. 1982. Recent trends in research on intestinal flora. Bifidobacteria Microflora. 1: 3-24; Salminen,S., Von-Wright,A., Morelli,L, Marteau,P., Brassart,D., des Vos,W., Fonden,R., Saxelin,M., Collins,K., Mogensen,G., Birkeland,S.E. and Mattila Sandholm,T. (1998). Demonstration of safety of probiotics - a review. Intl. J. Fd. Microbiol., 44(1-2): 93-106). It can also promote beneficial immune functions (Salminen,S., Isolauri,E., and Salminen, E. 1996. Clinical uses of probiotics for stabilizing the gut mucosal barrier: Successful

technological properties of a probiotic to be incorporated into food products is not to lose viability and functionality or creating unpleasant flavours or textures.
The viability and stability of probiotics have been both a marketing and
technological challenge for industrial producers. Probiotic foods should
contain food grade strain and maintain a suitable level of viable cells
during shelf-life of the products (Mattila,S.T., Blum,S., Collins,J.K.,
Crittenden,R., devos,W., Dunne,C, Fonden,R., Crenov,B., Isolauri,E.,
Kiely,B., Marteau,P., Morelli,L, Ouwehand,A., Reniero,R., Saarela,M.,
Salminen,S, Saxelin,M., Schiffrin,E., Shanahan,F., Vanghan,E., Von
and Wright,A. 1999. Probiotics: towards demonstrating efficacy.
Trends in Food Sci. and Technol., 10: 393-399). Pimia
R.Pupponen, Aura,A.M., Oksman,K.M., Caldentey,P., Myllarinen,M., Saarela., Sandholm,J.M. and Poutanen,K. 2002. Trends in Food Sci. Technol., 13: 3-11) have shown that the probiotic culture should have the properties to resist the gut environment. All lactic acid bacteria cannot be probiotic unless and until they fulfill the properties of resisting bile salt mix and low pH as that of the gut region. The culture strain should have a long exponential phase and be viable for a long period of time. For a probiotic curd, viability and stability of the

culture strain in the product is essential. This should also have a therapeutic minimum number of 105 cfu/g. To our knowledge, till date there is no probiotic dairy product in the Indian market. Also with the literature survey, it was found that the products sold abroad have either Lactobacillus or Lactococcus species cultures. With our work we have shown that the CFTRI culture isolate of Pediococcus pentosaceus MTCC 5151, which has been patented for probiotic activity has shown a good shelf life in curd and even after 21 days the number was in the range of therapeutic minimum as prescribed by WHO, and the sensory evaluation on hydonic scale showed that the product at 4°C and (-)20°C was fit to be consumed even after 21 days. This shows a good shelf life of the product.
Reference may be made to the work of Nighswonger,B.D., Brashears,M.M. and Gilliland,S.E. 1996. Survival of cells of Lactobacillus acidophilus and Lactobacillus casei during refrigerated storage in fermented milk products. J. Dairy Science, 79: 212-219, when they have studied the survival of Lactobacillus acidophilus and Lactobacillus casei during refrigerated storage in fermented milk products. They found that during the storage probiotic culture was viable and the traditional butter milk or yogurt cultures were not

recovered. The drawback is that the culture was similar as that present in the curd. For most of the potential health benefits to the consumer, it is necessary that the cells be viable when consumed. Typically these products are consumed within 2 to 3 weeks after production. Therefore, the cells should survive for at least three weeks to provide maximum health benefits to the consumers.
Reference may be made to the work of Bruno,E.A. and Shah,N.P. 2003. On the viability of two freeze dried strains of Bifidobacterium and of commercial preparations at various temperatures during prolonged storage. J. Food Sci. 68(7): 2336-2339, where they have worked out growth and viability of Bifidobacterium sp. in skim milk medium containing prebiotics. Usually the activity is lost but they found that addition of probiotic culture improved the retention of viability. Reference may be made to the work of Dave,R.I. and Shah, N.P. 1998. where Ingredient supplementation effects the viability of probiotic bacteria in yogurt. J. Dairy Sci., 81: 2804-2816 who studied the effect of cystein, whey powder, whey protein concentrate, acid casein hydroysates or tryptone on the viability of Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacteria. Here, they

found the viability with supplementation to be around 105 cfu/g till three week of storage.
A thorough patent search from 1976 till date has been made for 'probiotic curd' and 'probiotic yogurt'. Totally, sixty patents were found which covered probiotic therapy, their therapeutic and industrial applications. But, none of the patents deal with the supplementation of Pediococcus pentosaceus as a probiotic. The patents cover various species of Lactobacillus, Lactococci or Bifidobacterium. There is no data till now which gives probiotic therapy using Pediococcus pentosaceus. This makes the present work very important and novel and to be patented at the earliest.
The main objective of the present investigation is to formulate a probiotic curd and to study the stability and viability of the probiotic in the final product during storage.
The laboratory isolate MTCC 5151 was taxonomically identified as Pediococcus pentosaceus and earlier worked out in the laboratory as a potent probiotic culture was grown in MRS broth. After a period of 15 hr and after centrifugation, the cell pellet was reconstituted in10% skimmed milk to obtain a cell population of 2x109 cfu/g to be added to 100g curd. This served as a inocula in the market curd. The curd (100g) was mixed with a sterile spatula and dispensed into sterile container and stored at room temperature (RT),4°C and at(-)20°C.
Another object of the present investigation is to study the stability and viability of the probiotic culture in the product during storage for 21 days. Still another object of the present investigation was to find the storage time for a 'therapeutic minimum' of MTCC 5151 probiotic culture in the product. Still another objective of the present investigation was to find out if curd is a suitable carrier in order to keep the probiotic cells viable in the product. Still another objective is to formulate a shelf stable probiotic curd which is a healthful food for all ages and all strata of society. It has antimicrobial activity, can resist gut environment, is resistant to antibiotics which will help the patients to retain a gut microflora undergoing antibiotic treatment.
Statement of invention
Accordingly, the present invention provides a process for the preparation of shelf stable probiotic curd, which comprises the steps of: (a) inoculating curd characterized in that with a probiotic culture of Pediococcus pentosaceus having MTCC No. 5151 in
order to obtain an inoculated curd; (b) incubating the inoculated curd obtained in the step (a) at a temperature in the range of-28 to +28 degree Celsius for 21 days in order to obtain shelf stable probiotic curd having antimicrobial activity against Staphylococcus aureus; and (c) estimating the cell count and pH in the product.
In an embodiment of the present invention, the probiotic curd has all the above properties. To our knowledge there is no Pediococcus pentosaceus culture strain mentioned in the literature which is utilized in curd as a probiotic along with all the above properties. Therefore, there is a need to patent the information which is going to help the society at large.
In another embodiment of the present invention, as the culture is incorporated in curd, this food formulation will contribute towards better health and will help in rebalancing the digestive tract. As there is an interrelationship between intestinal microorganisms and health benefits, it is going to be of tremendous use. In recent years, this approach of incorporating probiotic bacteria into foods as dietary adjuncts for good health is on the forefront.
In another embodiment of the present invention, a
particular inoculum size of 2xl09 cfu/g when added is found to be optimum for good viability in the product.
In still another embodiment of the present invention, the
survival of probiotic culture strain after 7 days of storage was (4xl09) as much as that of zero period at RT and 4°C temperatures, but one log decline at (-) 20°C of storage conditions. As there is not much loss in viability which provides a high enough concentration of live probiotic is good for health benefits.
In still another embodiment of the present invention,
when the probiotic curd was stored for 14 days there was a decline of 3 logs at all the storage temperatures with maximum number (9xl06) at RT and minimum (5xl06) at (-) 20°C.
In still another embodiment of the present invention, on
storage for 21 days and studying the stability and viability of probiotic culture strain there was one more log decline at all temperatures in range of 2-3xl05. This number was still in the range of therapeutic minimum given in the literature.

In still another embodiment of the present invention, the
storage studies were done using lyophilized culture and reconstituting it in 10% sterilized skimmed milk. Freeze drying has long been the main process used to preserve probiotic organisms. Freeze drying is also used as a method to protect probiotics from starch acids and to deliver a high number of live organisms to the small and large intestines. Storage studies of probiotic curd for a period of 21 days show similar results as that of the fresh culture added.
NOVELTY
The novelty of the present work is that a locally isolated strain of Pediococcus pentosaceus MTCC 5151 being adopted for probiotic properties, is antimicrobial against Staphylococcus aureus, a toxic food pathogen and is resistant to antibiotics making it useful to patients under antibiotic treatment to maintain the gut microflora and when incorporated in curd showed a good shelf life till 21 days with a number prescribed as therapeutic minimum by WHO. This makes it a novel healthful food product for consumers of all strata and ages. The novel inventive step is adoption of native culture MTCC 5151 till date

with probiotic properties and the formation of 'probiotic' curd which is not known till date.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
The culture is a local isolate from curd and characterized as Pediococcus pentosaceus according to the Bergy's Manual of Systematic Bacteriology. The culture produced under stressed conditions was found to be antimicrobial against Staphylococcus aureus a toxic food pathogen. It can tolerate a low pH of 2.8 and bile acid mix upto 0.2%. The culture strain is resistant to Ox-gall upto 0.2% and is resistant to antibiotics.
EXAMPLE 1
This example gives the addition of culture inoculum to curd sample. The culture used in the experiment to test the stability of the probiotic culture was prepared as such in MRS broth or lyophilized. Initially plating was done by taking the cells and reconstituting it in 10% skimmed milk. Inoculum size of 3xl09 to 2xl09 cfu/g viable cells were

found to be the best to be used as an inoculum. Inoculation was done into the fermented product (100g) in pre-sterilized cups and stirred with a sterile glass rod for homogenous mixing of probiotic inoculum. The cups were stored at RT, 4°C and at (-) 20°C for studying storage studies.
Every week, all samples were taken for plating in selective media for cell count and pH analysis.
Table 1: Analysis of initial inoculum cell count and curd
(Table Removed)

EXAMPLE 2
This example gives the stability and viability of probiotic culture strain MTCC 5151 in curd after 7 days. After seven days, the aliquot was taken out and different parameters were studied. For enumeration of

the plate count of probiotic culture the plating was done on selective media (MRSA with 6.5% NaCI and MRSA (9.6 pH). For total counts, the sample aliquot was plated on MRSA media. As can be seen from the table, the probiotic culture showed excellent stability and viability at RT and at 4°C although there was one log decrease at -20°C. Each time, the curd sample alone (without the probiotic culture) was taken as control.
Table 2: Stability and viability of probiotic culture in curd after 7 days

(Table Removed)
count was made on all different media for cfu/g and it was found that the probiotic count declined by three logs at RT and at 4°C and two logs at (-) 20°C from that of 7 days. The cell counts, flavour and pH were in the range of therapeutic minimum and taste was good at all temperatures.
Table 3: Stability and viability of probiotic culture in curd after 14 days
(Table Removed)

EXAMPLE 4
This example shows the results after 21 days. The colonies were counted by plating on all different media and it was seen that at all

the three temperatures, there was one more log decline from 14 days. The sample kept at RT was not palatable although the samples kept at 4°C and (-) 20°C were found good. Even though, there was a decline in cell count still the viable counts in the final product were in the range of therapeutic minimum and sufficient for health benefits.
Table 4: Stability and viability of probiotic culture in curd after 21 days
(Table Removed)

EXAMPLE 5
This example gives the stability and viability of probiotic culture in curd after 28 days. The good shelf life of P. pentosaceus in curd shows that it is a suitable carrier and emphasizes the importance of this particular culture in the preparation of a novel probiotic curd to provide potential health benefits to the consumers. It is an important technological quality of a probiotic containing product that they support the survival of the probiotic.
Table 5: Stability and viability of probiotic culture in curd after 28 days
(Table Removed)

THE novelty of the present invention is "A process for production of shelf stable probiotic curd" with a good storage life and beneficial health improvement.
The main advantages of the present investigation are:
1. The isolate is a food grade bacteria isolated from the cheese
2. A novel probiotic curd for health benefits to the consumers is produced
3. The storage life of probiotic culture for 21 days shows the stability of the culture strain
4. The culture strain is resistant to low pH and bile salts, making it suitable to remain in gut environment for a longer time
5. The viability in curd has shown that it is a good carrier for the culture
6. Even after 21 days of storage and decline in number of cells, still it is in the range of therapeutic minimum. For the first time a process for production of shelf stable probiotic curd with Pediococcus pentosaceus is reported.

We claim:
1) A process for the preparation of shelf stable probiotic curd,
which comprises the steps of: (a) inoculating curd characterized in that with a probiotic culture of Pediococcus pentosaceus having MTCC No. 5151 in order to obtain an inoculated curd; (b) incubating the inoculated curd obtained in the step (a) at a temperature in the range of -28 to +28 degree Celsius for 21 days in order to obtain shelf stable probiotic curd having antimicrobial activity against Staphylococcus aureus; and (c) estimating the cell count and pH in the product.
2) A process as claimed in claim 1, wherein the probiotic inoculum used is added at a concentration of about 2 X 109 cfu (colony forming units ) per milliliter to curd sample.
3) A process as claimed in claims 1 and 2, wherein the incubation used is at a temperature preferably in the range of 0 degree Celsius to minus 20 degree Celsius.

4) A process as claimed in claims 1-3, wherein the cell count used was studied in the sample every week by plating on de Man Rogosa and Sharpe medium.
5) A process as claimed in claims 1-4. wherein the shelf stable probiotic curd obtained has the pH in the range of 4.6 -4.8.
6) A process as claimed in claims 1-5, wherein the shelf stable probiotic curd obtained has a cell count in the range 1.5 X 105 to 3 X 105 cfu ( colony forming units ) per milliliter of therapeutic minimum given by WHO.
7) A process for preparation of shelf stable probiotic curd substantially as
herein described in description and exemplified with accompanying
examples.